A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue

A microscale procedure for the isolation and purification of viroid RNA from one gram of viroid-infected leaf tissue and for its subsequent sequencing at the cDNA level is described using potato spindle tuber viroid (PSTV) as model system. Total nucleic acids are phenol-extracted and salt-fractionated with 2 M LiCl. The viroid-containing fraction is then subjected to bidirectional polyacrylamide gel electrophoresis. This removes all co-fractionated cellular RNAs from the circular viroid RNA which is finally recovered from the gel in pure form by isotachophoresis. Thus, from one gram of PSTV-infected tomato leaf tissue, about 100-250 ng of circular PSTV RNA can be obtained and used as template for several DNA primer-directed reverse transcription reactions. From the primer-extended overlapping cDNAs the entire sequence of the viroid progeny synthesized in an individual plant or plant leaf can thus be established by Maxam-Gilbert sequencing. This renders the procedure especially suited for the routine analysis of the in vivo fate of viroid mutants constructed in vitro.